ESI-LC-MS/MS Measurements of Human Urine Standard Reference Materials

The NIST Mass Spectrometry Data Center has been performing the metabolite profiling of several standard reference materials (SRM) from biofluids, such as human blood and urine, using hyphenated mass spectrometry techniques. Our focus has been primarily on developing applications for the NIST Mass Spectral Libraries to facilitate their use and reach specific scientific communities. However, these datasets can be useful in bioinformatics, AI, clinical applications, and more. Here, we include the urine raw data and associated metadata.
Materials and Methods
Reagents: HPLC-grade methanol and water were obtained from Honeywell, Burdick & Jackson. HPLC-grade acetonitrile was purchased from EMD Millipore, and LC-MS-grade formic acid was obtained from Fluka. Creatinine-(N-methyl-d3) (creatinine-d3; 98% purity), used as the internal standard for semi-quantitative analysis, was purchased from Sigma-Aldrich.
Samples: Six NIST SRMs) urines represent donors with differing smoking status and exposure histories, including non-smokers, passive smokers, and active smokers as follows: SRMs: 3667, pooled urine from ≥10 donors, both male and female [1], 3671-1, pooled urine from ≥10 non-smoking donors without exposure to tobacco smoke [2], 3671-2, pooled urine from ≥10 non-smoking donors with exposure to secondhand smoke [2], 3671-3, pooled urine from ≥10 donors who smoked at least one pack of cigarettes per day [2], 3672 pooled urine from donors who smoked more than one pack of cigarettes per day [3, 4] and 3673, pooled urine from non-smoking donors not exposed to secondhand smoke [3, 5].
Sample prep: Urine samples (SRMs) were thawed from -80 °C and kept at room temperature for 3 h. Each urine sample (300 µL) was collected in an Eppendorf tube, and 300 µL of methanol was added. The samples were centrifuged (Beckman Coulter Centrifuge Microfuge 22R) for 20 min at 10000g and room temperature. The supernatant was evaporated to dryness for 90 min under a nitrogen stream at room temperature. The dried material was reconstituted with 200 µL of a water solution of the internal standard creatinine-d3 (320 ng/µL). The final 200 µL solution was processed from 300 µL urine sample. The samples were maintained at 4 °C in the auto-sampler and analyzed by LC-MS.
ESI-LC-MS/MS analysis: Reverse phase separations were accomplished with the use of a Dionex Ultimate 3000 system (Dionex, USA) with 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). A Waters ACQUITY UPLC CSH-C18 Column, 130 Å (2.1x100 mm, 1.7 µm) was used at 500 to 600 bar. A gradient [time (min), %B]: (0, 1) (1, 1) (18, 20) (24, 90) (27, 90) (28, 1) (30, 1) was completed at 30 min. The injection volume was 3 µL, and the flow rate was 0.4 mL/min.
Mass spectra were recorded in positive-ionization mode, vaporizer temperature 300 °C, spray voltage 3500 V, S-Len RF level 50, sheath gas (Arb) 45, AUX gas (Arb) 13, sweep gas (Arb) 1 to obtain maximum abundance of the standard sample in full-scan spectra (80 Da to 1200 Da) using an Orbitrap Fusion Lumos. The resolution for the full MS scan was set to 120,000, and for the MS/MS scan to 30,000. Data-dependent acquisition (DDA) was performed with an exclusion duration of 18 s and an isolation window of ±0.75 m/z. Duplicate injections were performed for each of the six samples at each of the nine normalized collision energies (NCEs: 10, 15, 20, 25, 30, 40, 50, 60, and 80), resulting in a total of 108 runs (6 samples × 9 NCEs × 2 runs/sample/NCE).